The present invention is directed to novel achiral seco-analogues of (+)-CC1065 and the duocarmycins and pharmaceutical compositions containing the said achiral analogues. The achiral analogues of (+)-CC1065 and the duocarmycins are useful as anticancer agents.
One class of compounds that has received much attention recently is the DNA minor groove binders that exert their anticancer activity by alkylating specific sequences of DNA (1a). Minor groove interacting agents are more attractive than intercalating agents and major groove binders because the minor groove is typically only occupied by a spine of hydration and is therefore more accessible to anticancer agents. Additionally, covalent modifications in the minor groove are generally more cytotoxic to cells than their major groove alkylating counterparts, such as tallimustine versus L-phenylalanine mustard (1b). Examples of minor groove and AT sequence selective alkylating agents that have potent anticancer activity are (+)-CC-1065 (1c,d,e, 2) and the duocarmycins (2, 3). CC-1065 and the duocarmycins, exemplified by (+)-duocarmycin SA (or DUMSA) (2) and depicted in Table 1, belong to a group of natural products that have incredibly potent cytotoxic properties (ca. IC50 values in the pM range against the growth of mouse L1210 leukemia cells in 
Table 1. Structures and summaries of the biological properties of (+)-CC1065 and doucarmycin SA (DUMSA) culture). These compounds, which were isolated from the fermentation broth of Streptomyces zelensis (4) and Streptomryces sp. (5), respectively, derive their anticancer activity by reacting primarily with adenine-N3 groups in the minor groove of specific sequences of DNA. Confirmation of the adenine-N3 covalent reaction was achieved from isolation of an adenine-CC1065 adduct (6) and an adenine-duocarmycin SA adduct (7) from thermally cleaved DNA-drug adducts. (+)-CC1065 shows a preference for alkylating the adenine-N3 group which is flanked by 5xe2x80x2-A or 5xe2x80x2-T residues. The sequence preference for the three-base AT-rich alkylation site followed the order: 5xe2x80x2-AAA=5xe2x80x2-TTA greater than 5xe2x80x2-TAA greater than 5xe2x80x2-ATA (the alkylation site is denoted by the underlined base). In addition, this compound exhibited a strong preference for the fourth 5xe2x80x2-base to be an A or T residue, a weaker preference for the fifth 5xe2x80x2-base to be an A or T base, and a weak preference for the 3xe2x80x2 base preceding the alkylating site to be a purine. The consensus sequences 5xe2x80x2-PuNTTA-3xe2x80x2 and 5xe2x80x2-AAAAA-3xe2x80x2 for (+)-CC-1065 are suggested (6, 8). Although (+)-CC1065 and (+)-DUMSA generally have similar sequence selectivity, some subtle differences were observed, notably the lack of alkylation at 5xe2x80x2-GCAAA by CC1065 (2, 3, 7).
The mechanism by which CC1065 and the duocarmycins react with specific sequences of DNA is still a subject of intense debate (9). Two models for their sequence selective DNA alkylations have been proposed. The xe2x80x9cnoncovalent binding modelxe2x80x9d proposed by Boger""s group (9) suggests that the forces stabilizing the DNA alkylation are a combination of stereoelectronically controlled covalent bond formation between the cyclopropylpyrroloindolone (CPI) subunit of CC1065 and adenine-N3 of DNA, as well as stabilizing noncovalent interactions derived from the hydrophobic and van der Waals contacts of the PDE-I subunits for CC1065, and the TMI (trimethoxyindole) unit of DUMSA (9). In the xe2x80x9calkylation site modelxe2x80x9d proposed by Hurley and coworkers (10), the covalent sequence specificty of the compounds, exemplified by (+)-CC1065, has been attributed to the unique conformational features of the consensus sequences, in which the sequences are flexible enough to adopt a transient bent conformation for recognition of the drug molecule. Further, the unique conformation provides an acidic proton on a phosphate which can activate the CPI system by general acid catalysis. In either case, 1H-NMR studies showed that (+)-CC1065 effectively alkylated adenine-N3 of duplex 5xe2x80x2-GGCGGAGTTAGG-3xe2x80x2 (alkylation at the underlined A residue in the italicized sequence) and induced a bend of 17-22 ▭ at the TTA sequence (11). Similarly, a 1H-NMR study of DUMSA with d-(GACTMTTGAC).d-(GTCATTAGTC) has also revealed that the drug undergoes significant conformational changes upon binding to the minor groove, which subsequently enhances its covalent reactivity (lid). Such conformational changes on the DNA has been postulated to entrap structures in the DNA that might be relevant to the biological control of gene expression and replication (12). A recent density functional and ab Initio study on the activation of the duocarmycin SA pharmacophore for DNA alkylation was reported (13). In the study the authors found that twisting of the indoline-amido-N bond (▭2) did not sufficiently explain the million-fold enhancement of DNA alkylation compared to solvolysis.
Even though (+)-CC1065 has potent cytotoxic properties, its usefulness as an anticancer drug is hampered by its limiting toxicity of delayed lethality in mice at therapeutic doses (4b). Interestingly, DUMSA is devoid of this toxic side effect (2a), and it is the most solvolytically stable and the most potent in this class of compounds (2, 14). Facilitated by an extensive array of analogues of CC1065 synthesized by Upjohn scientists (1a, b, 4c), structure-activity relationships have been sharply defined (10b, 11), and the ethano bridges (see FIG. 1) were found to be responsible for the undesired delayed lethality. These ethano groups enter into favorable van der Waals and hydrophobic interactions with adenine-H2 atoms on the floor of the minor groove that stabilizes the drug-DNA complex (11a, b). It has been suggested that the delayed lethality of (+)-CC1065 and its CDPI2 analogue (see Table 2) is a consequence of their inability to xe2x80x9creversiblyxe2x80x9d alkylate DNA (7, 15). The strong noncovalent binding of these compounds due to the ethano groups could either prevent the reverse reaction after alkylation has occurred (7, 15a), or keeps the drug bound so that it realkylates the same site (15b). In agreement with this suggestion, CC1065 analogues, such as adozelesin (15b) and DUMSA (15a, 16), which lack the ethano bridges, are devoid of the delayed toxicity problem, yet they exhibit potent anticancer activity. 
Adozelesin (1b, 4c, 17, patent 1), carzelesin (18), bizelesin (19, patent 2), and KW2189 (20, patent 3) are examples of analogues of (+)-CC1065 and duocarmycins that are presently undergoing clinical trials for the treatment of cancer. A phase I clinical trial of adozelesin has shown a partial response on a melanoma patient (17e). Carzelesin is a prodrug that upon carbamate hydrolysis provides the seco-prodrug U76073, which readily cyclizes to the corresponding cyclopropane containing CPI xe2x80x9cdrugxe2x80x9d U76074 (18). It is worthy to note that carzelesin has a higher in-vivo anticancer activity against L1210 leukemia than adozelesin (150xc2x18% ILS, 2/6 survival versus 90xc2x111%, 0/6 survival) (18). This outcome is likely to be due to the lower toxicity of carzelesin, and consequently it can be administered at a higher dose (400 xcexcg/kg vs 100 xcexcg/kg for adozelesin) (18). Bizelesin is a DNA interstrand crosslinking agent incorporating two seco-CPI alkylating units and is approximately 20-30 times more potent than (+)-CC1065 itself (IC50 (L1210)=1 pM vs 20 pM) (19). KW2189, a semisynthetic duocarmycin B2 derivative, which possesses improved anticancer activity, water solubility, and stability, has been selected for clinical evaluation in Japan (20). Its mechanism of activation also involves hydrolysis of the carbamate group to release a seco-prodrug, which cyclizes to produce the actual cyclopropane containing drug, or DU-86.
The biological properties of these clinically studied compounds indicate that seco-prodrugs are as active as their cyclopropane containing CPI counterparts but do not cause the delayed toxicity shown by (+)-CC1065. Because seco-prodrugs are also more stable and easier to handle than the drugs themselves, compounds bearing this type of pharmacophore are described in this disclosure. In addition, the indole-benzofuran (In-Bf) (4c), the indole-indole (In-In) (4c), the trimethoxyindole (TMI) (3), as well as analogues of distamycin as exemplified by the dipyrrole (Py-Py) (21) noncovalent binding units given in Table 2 will be incorporated into the structures of the compounds of the invention. These DNA binding units are chosen because they have been shown to enhance the cytotoxic potency of this class of compounds, and they lack the delayed toxicity problem.
Besides systematic modifications on the noncovalent portion of CC1065, alterations of the pharmacophore have also been done to elucidate the mechanism by which this class of compounds exert their activities. Analogues of the CPI unit of CC1065 and duocarmycin SA (DUMSA) (1-4) depicted in Table 2, such as CBI (cyclopropylbenzoindolone) (2, 3, 22), CFI (cyclopropylfuranoindolone) (23), CPzI (cyclopropylpyrazoloindolone) (24) and CI (cyclopropylindolone) (2, 3, 25), have been designed and prepared. Structure-activity relationship studies of these pharmacophores have demonstrated that analogues possessing the greatest solvolytic stability also exhibited the most potent anticancer activity (9b, 14). Moreover, amino-containing seco-CI and CBI analogues, depicted in Table 3, have also been shown to be equally potent in inhibiting the growth of cancer cells as the parent seco-CI and CBI compounds (26). 
Effects of the chiral center on the biological activity of this class of compounds have also been studied. The unnatural (xe2x88x92)-(R) enantiomer of DUMSA was ten times less effective in alkylating DNA than the natural (+)-(S) enantiomer, and that was consistent with the ten fold lower in cytotoxicity for the (xe2x88x92)-enantiomer (2, 7). Similar to (+)-CC1065, the binding orientation of (+)-DUMSA is 3xe2x80x2- greater than 5xe2x80x2 over an AT-rich 3-5 base pair site, and the opposite enantiomer orients in the 5xe2x80x2- greater than 3xe2x80x2 direction. It is worth noting that while (xe2x88x92)-CC1065 exhibited a slower rate of DNA alkylation than the (+)-enantiomer, their overall efficiencies of DNA alkylation are identical, and so are their in-vitro and in-vivo anticancer activities (27). Further investigations on both enantiomers of CPI-CDPI1 and CPI-PDE-I1 (see Table 2 for general structures) revealed their cytotoxicities differ by a factor of about 150 fold favoring the natural (+)-isomer (2a, 27b). For the enantiomers of CPI-In-In, the natural (+)-isomer is about 250 times more cytotoxic (27c).
It is, therefore, crucial that enantiomerically pure compounds must be prepared and evaluated biologically before they can be developed into clinical drugs (28a). At present, syntheses of this class of compounds in the optically pure forms require a chemical resolution step, either by separation on a chiral column by HPLC (29) or separation of diastereomeric Mandelate (2, 21, 30a,b) or N-BOC-L-tryptophan esters (30c) of intermediates using normal phase HPLC and recrystallization methods, respectively. There is also a report of using lipase catalyzed esterification reactions to resolve racemic alcohol intermediates for synthesizing optically active CPI (31). While these methods are feasible, at least in small scale, they are inefficient because in the chemical resolution step all of the undesired enantiomer has to be discarded. Furthermore, there have been reports of only modest asymmetric induction in the preparation of CFI (23), CBI (32) and duocarmycin A (29), using either an asymmetric hydroboration or Sharpless dihydroxylation method, respectively.
The achiral analogues of CC-1065 and doucarmycins described in this invention will thus represent a significant improvement to the present state in the development of this class of minor groove binding agents for use in the treatment of diseases such as cancers. Synthesis of the achiral analogues described in this disclosure will not only alleviate the chemical resolution step, their interactions with DNA may be simplified, and their cytotoxic potency and anticancer activity are retained. The design of the target compounds described in this invention is based on a report that 4-(2-haloethyl)phenols (halide=Cl, Br and I) are capable of producing DNA alkylation and cytotoxic activities (28b). 4-(2-Haloethyl)phenols have also been shown to readily eliminate HX to generate a spiro(2,5)octa-1,4-diene-3-one intermediate which ultimately alkylates DNA (28c).
In this disclosure, the preparation and evaluation of the biological and DNA interacting properties of a series of achiral seco analogues of the doucarmycins, CC-1065 and adozelesin are described. The structures of the seco-achiral compounds of this invention are depicted as general class I, II, III and IV. The achiral compounds are composed of an achiral pharmacophore conjugated to a number of non-covalent DNA binding subunit, including, but not limited to, the Indole-Indole, Indole-Benzofuran, Pyrrole-Pyrrole, Trimethoxyindole, the N-mustard (H) as well as the xe2x80x98hairpinxe2x80x99 (J) groups as depicted in Table 4. The xe2x80x98hairpinxe2x80x99 subunit was designed on the basis of recent findings that 1:1 and side-by-side complexes of duocarmycin A and distamycin (33a,b) or lexitropsins (33c,d) exhibited a high degree of DNA sequence alkylation specificity. In addition, xe2x80x98hairpinxe2x80x99 duocarmycin-oligopeptides conjugates that are related to distamycin and lexitropsins have been shown to exhibit a predictable and high level of sequence specificity (34). Yet in addition, side-by-side and hairpin oligopeptides derived from the lexitropsins have been extensively studied. The studies revealed that the side-by-side pyrrole-pyrrole pairing recognizes either an A/T or T/A base pair. A pyrrole-imidazole pairing specifically recognizes a C/G base pair, while an imidazole-pyrrole pair binds specificalaly to a G/C base pair, respectively (35). Recently we have also demonstrated that the imidazole-imidazole pairing will recognize either a G/C or C/G within specific DNA sequences (36). Moreover, the side-by-side imnidazole-imidazole pairing was found to be even more selective for binding to mismatched T/G base pairs, providing a unique opportunity to develop molecules for targeting such mismatched DNA base pairs (37).
The present invention relates to novel achiral analogues of the DNA minor groove alkylating agents (+)-CC1065 and the duocarmycins, depicted as general class I, II, III, IV, and V: 
wherein X is a good leaving group, such as a chloride, a bromide, an iodide, a mesylate, a tosylate, an acetate, a quaternary ammonium moiety, a mercaptan, an alkylsulfoxyl, or an alkylsulfonyl group, preferably either a chloride, a bromide, or an iodide group.
R1 is a suitable minor groove binding agent to enhance the interactions of the achiral seco-cyclopropaneindole (CI) or an achiral seco-duocarmycin with specific sequences of DNA. Examples of the DNA binders are given in Table 4. The preferred DNA binders are groups A, C, D, E, F, G, H, I, J, K and L. R1 can also include the following: t-butoxy, benzyloxy, 9-fluorenylmethyloxy or other common protecting groups for amines. 
R2 and R3 can be hydrogen or short chain alkyl (C1-C5) groups, preferably both being hydrogen atoms. The alkyl groups may be straight chain or branched and include such groups as ethyl, propyl, butyl, pentyl and hexyl.
R4 and R5 can be hydrogen atoms, short alkyl groups, trifluoromethyl mojeties, and alkyloxycarbonyl groups. The preferred R4 and R5 groups are methoxycarbonyl and trifluoromethyl.
R can be either a benzyl, a benzyloxycarbonyl, a hydrogen atom, a 4-nitrobenzyloxycarbonyl, or a Nxe2x80x2-methylpiperazinyl-N-carbonyl group.
Another aspect of the present invention is directed to a method for the treatment of cancer by administering the compounds of the present invention to a patient for a time and under conditions sufficient to effect inhibition of cancerous growth.
Another aspect of the present invention is directed to a method for the rcognition and targeting of compounds described in the invention to specific DNA sequences, including sequences within the control regions of disease causing genes. Such agents can have application for the identification of unique sequences in the genome as well as for use as potential gene controlling properties (38).
Yet another aspect of the present invention provides pharmaceutical compositions containing the compounds of the invention in a combination with pharmaceutical carriers.